Photoreceptor signaling in vertebrate rods and cones consists of a series of precisely timed events that are critical for photoreceptors to function under a broad range of light intensities. While rods operate under dim light and are easily saturated in response to bright light, cones are less sensitive to light, recover more rapidly and are able to function under intense light. G protein-coupled receptor kinases (GRKs) in the vertebrate retina initiate turnoff of the photoresponse in both rods and cones via phosphorylation of rhodopsin and the cone opsins, respectively. We have discovered that many vertebrate species, including humans, express both GRK1 and GRK7 in cones, raising the question: do these kinases have distinct or overlapping functions in the photoresponse and adaptation? We have also shown that both kinases are phosphorylated by cAMP-dependent protein kinase (PKA) in dark-adapted animals and dephosphorylated in light-adapted animals. Phosphorylation reduces the activity of these GRKs in vitro. Therefore we propose a novel hypothesis that cAMP plays an important regulatory role in phototransduction and/or adaptation through its downstream kinase, PKA. We have generated model animals to (a) determine the distinct or overlapping roles played by GRK1 and GRK7 in cones in vivo and (b) test the hypothesis that cAMP-mediated phosphorylation of these kinases in rods and cones plays an important role in photoreceptor signaling. Specific Aim 1 addresses the contributions of GRK1 and GRK7 to the cone photoresponse and adaptation using genetically modified zebrafish. Unlike mice, zebrafish express both GRK1 and GRK7 in cones, similar to humans. Therefore zebrafish is the best genetic model for studies of cones. We used TALENs to disrupt the genes for grk1a and grk7b to create null mutants. Analyzing these lines by electroretinography (ERG) in 5 dpf zebrafish will allow us to define the contribution of each kinase to cone signaling. At this stage of development, the zebrafish retina is functionally an all cones retina. A light intensity/response series and paired flash experiments with or without background light will be used to determine the effect of deleting these kinases individually on the kinetics of the photoresponse and adaptation. Specific Aim 2 addresses the role of phosphorylation of GRK1 and GRK7 by PKA in zebrafish cones. We have generated lines expressing the phosphorylation-site mutants, Grk1b-S21A, Grk1b-S21E, Grk7a-S33A and Grk7a-S33E. These mutant zebrafish lines will be crossed with the knockout lines and evaluated using experiments similar to those described for Specific Aim 1. Specific Aim 3 addresses the influence of phosphorylation of GRK1 by PKA in both rods and cones in mice where mutations S21A and S21E have been knocked into the Grk1 gene. These genetically modified mice express levels of mutant GRK1 that are identical to the wild type protein. Suction electrode recording and ERG will be performed to define the role of phosphorylation of GRK1 on photoreceptor signaling in rods and cones in mice.